A thermosensitive PCNA allele underlies an ataxia-telangiectasia-like disorder.

Proliferating cell nuclear antigen (PCNA) is a sliding clamp protein that coordinates DNA replication with various DNA maintenance events that are critical for human health. Recently, a hypomorphic homozygous serine to isoleucine (S228I) substitution in PCNA was described to underlie a rare DNA repair disorder known as PCNA-associated DNA repair disorder (PARD). PARD symptoms range from UV sensitivity, neurodegeneration, telangiectasia, and premature aging. We, and others, previously showed that the S228I variant changes the protein-binding pocket of PCNA to a conformation that impairs interactions with specific partners. Here, we report a second PCNA substitution (C148S) that also causes PARD. Unlike PCNA-S228I, PCNA-C148S has WT-like structure and affinity toward partners. In contrast, both disease-associated variants possess a thermostability defect. Furthermore, patient-derived cells homozygous for the C148S allele exhibit low levels of chromatin-bound PCNA and display temperature-dependent phenotypes. The stability defect of both PARD variants indicates that PCNA levels are likely an important driver of PARD disease. These results significantly advance our understanding of PARD and will likely stimulate additional work focused on clinical, diagnostic, and therapeutic aspects of this severe disease.

High-throughput splicing assays identify missense and silent splice-disruptive POU1F1 variants underlying pituitary hormone deficiency.

Pituitary hormone deficiency occurs in ∼1:4,000 live births. Approximately 3% of the cases are due to mutations in the alpha isoform of POU1F1, a pituitary-specific transcriptional activator. We found four separate heterozygous missense variants in unrelated individuals with hypopituitarism that were predicted to affect a minor isoform, POU1F1 beta, which can act as a transcriptional repressor. These variants retain repressor activity, but they shift splicing to favor the expression of the beta isoform, resulting in dominant-negative loss of function. Using a high-throughput splicing reporter assay, we tested 1,070 single-nucleotide variants in POU1F1. We identified 96 splice-disruptive variants, including 14 synonymous variants. In separate cohorts, we found two additional synonymous variants nominated by this screen that co-segregate with hypopituitarism. This study underlines the importance of evaluating the impact of variants on splicing and provides a catalog for interpretation of variants of unknown significance in POU1F1.

SCUBE3 loss-of-function causes a recognizable recessive developmental disorder due to defective bone morphogenetic protein signaling.

Signal peptide-CUB-EGF domain-containing protein 3 (SCUBE3) is a member of a small family of multifunctional cell surface-anchored glycoproteins functioning as co-receptors for a variety of growth factors. Here we report that bi-allelic inactivating variants in SCUBE3 have pleiotropic consequences on development and cause a previously unrecognized syndromic disorder. Eighteen affected individuals from nine unrelated families showed a consistent phenotype characterized by reduced growth, skeletal features, distinctive craniofacial appearance, and dental anomalies. In vitro functional validation studies demonstrated a variable impact of disease-causing variants on transcript processing, protein secretion and function, and their dysregulating effect on bone morphogenetic protein (BMP) signaling. We show that SCUBE3 acts as a BMP2/BMP4 co-receptor, recruits the BMP receptor complexes into raft microdomains, and positively modulates signaling possibly by augmenting the specific interactions between BMPs and BMP type I receptors. Scube3-/- mice showed craniofacial and dental defects, reduced body size, and defective endochondral bone growth due to impaired BMP-mediated chondrogenesis and osteogenesis, recapitulating the human disorder. Our findings identify a human disease caused by defective function of a member of the SCUBE family, and link SCUBE3 to processes controlling growth, morphogenesis, and bone and teeth development through modulation of BMP signaling.

Exome Sequencing Identifies Multiple Genetic Diagnoses in Children with Syndromic Growth Disorders.

OBJECTIVE: To evaluate the presence of multiple genetic diagnoses in syndromic growth disorders. STUDY DESIGN: We carried out a cross-sectional study to evaluate 115 patients with syndromic tall (n = 24) or short stature (n = 91) of unknown cause from a tertiary referral center for growth disorders. Exome sequencing was performed to assess germline single nucleotide, InDel, and copy number variants. All variants were classified according to ACMG/AMP guidelines. The main outcome measured was the frequency of multiple genetic diagnoses in a cohort of children with syndromic growth disorders. RESULTS: The total diagnostic yield of the cohort was 54.8% (63/115). Six patients had multiple genetic diagnoses (tall stature group = 2; short stature group = 4). The proportion of multiple diagnoses within total cases was 5.2% (6/115), and within solved cases was 9.5% (6/63). No characteristics were significantly more frequent when compared with patients with single or multiple genetic findings. Among patients with multiple diagnoses, 3 had syndromes with overlapping clinical features, and the others had syndromes with distinct phenotypes. CONCLUSION: Recognition of multiple genetic diagnoses as a possibility in complex cases of syndromic growth disorders opens a new perspective on treatment and genetic counseling for affected patients, defying the medical common sense of trying to fit all findings into one diagnosis.

Diagnosis of TBC1D32-associated conditions: Expanding the phenotypic spectrum of a complex ciliopathy.

Exome sequencing is a powerful tool in prenatal and postnatal genetics and can help identify novel candidate genes critical to human development. We describe seven unpublished probands with rare likely pathogenic variants or variants of uncertain significance that segregate with recessive disease in TBC1D32, including four fetal probands in three unrelated pedigrees and three pediatric probands in unrelated pedigrees. We also report clinical comparisons with seven previously published patients. Index probands were identified through an ongoing prenatal exome sequencing study and through an online data sharing platform (Gene Matcher™). A literature review was also completed. TBC1D32 is involved in the development and function of cilia and is expressed in the developing hypothalamus and pituitary gland. We provide additional data to expand the phenotype correlated with TBC1D32 variants, including a severe prenatal phenotype associated with life-limiting congenital anomalies.

SIN3A Defects Associated with Syndromic Congenital Hypogonadotropic Hypogonadism: An Overlap with Witteveen-Kolk Syndrome.

INTRODUCTION: Congenital hypogonadotropic hypogonadism (CHH) is a rare condition caused by GnRH deficiency. More than 40 genes have been associated with the pathogenesis of CHH, but most cases still remain without a molecular diagnosis. Mutations involving the same gene (e.g., FGFR1, PROK2/PROKR2, CHD7) were found to cause normosmic CHH and Kallmann syndrome (KS), with and without associated phenotypes, illustrating the coexistence of CHH with signs of other complex syndromes. The Witteveen-Kolk syndrome (WITKOS), caused by defects of the SIN3A gene, is a heterogeneous disorder characterized by distinctive facial features, microcephaly, short stature, delayed cognitive, and motor development. Although micropenis and cryptorchidism have been reported in this syndrome, WITKOS has not been formally associated with CHH so far. PATIENTS AND METHODS: A man with KS associated with mild syndromic features (S1) and a boy with global developmental delay, syndromic short stature, micropenis and cryptorchidism (S2), in whom common genetic defects associated with CHH and short stature had been previously excluded, were studied by either chromosomal microarray analysis or whole exome sequencing. RESULTS: Rare SIN3A pathogenic variants were identified in these 2 unrelated patients with CHH phenotypic features. A 550 kb deletion at 15q24.1, including the whole SIN3A gene, was identified in S1, and a SIN3A nonsense rare variant (p.Arg471*) was detected in S2. CONCLUSION: These findings lead us to propose a link between SIN3A defects and CHH, especially in syndromic cases, based on these 2 patients with overlapping phenotypes of WITKOS and CHH.

Phenotypic and mutational spectrum of ROR2-related Robinow syndrome.

Robinow syndrome is characterized by a triad of craniofacial dysmorphisms, disproportionate-limb short stature, and genital hypoplasia. A significant degree of phenotypic variability seems to correlate with different genes/loci. Disturbances of the noncanonical WNT-pathway have been identified as the main cause of the syndrome. Biallelic variants in ROR2 cause an autosomal recessive form of the syndrome with distinctive skeletal findings. Twenty-two patients with a clinical diagnosis of autosomal recessive Robinow syndrome were screened for variants in ROR2 using multiple molecular approaches. We identified 25 putatively pathogenic ROR2 variants, 16 novel, including single nucleotide variants and exonic deletions. Detailed phenotypic analyses revealed that all subjects presented with a prominent forehead, hypertelorism, short nose, abnormality of the nasal tip, brachydactyly, mesomelic limb shortening, short stature, and genital hypoplasia in male patients. A total of 19 clinical features were present in more than 75% of the subjects, thus pointing to an overall uniformity of the phenotype. Disease-causing variants in ROR2, contribute to a clinically recognizable autosomal recessive trait phenotype with multiple skeletal defects. A comprehensive quantitative clinical evaluation of this cohort delineated the phenotypic spectrum of ROR2-related Robinow syndrome. The identification of exonic deletion variant alleles further supports the contention of a loss-of-function mechanism in the etiology of the syndrome.

Genetic investigation of syndromic forms of obesity.

BACKGROUND: Syndromic obesity (SO) refers to obesity with additional phenotypes, including intellectual disability (ID)/developmental delay (DD), dysmorphic features, or organ-specific abnormalities. SO is rare, has high phenotypic variability, and frequently follows a monogenic pattern of inheritance. However, the genetic etiology of most cases of SO has not been elucidated. SUBJECTS AND METHODS: In this study, we investigated 20 SO patients by whole-exome sequencing (WES) trios to identify causal genetic variants. RESULTS: 4/20 patients had negative results for array comparative genomic hybridization (aCGH) analyses. In the remaining 15 patients, in addition to SNVs and indels, CNVs were also evaluated. Pathogenic/likely pathogenic (P/LP) SNVs/indels were detected in 6/20 patients (involving MED13L, AHDC1, EHMT1, MYT1L, GRIA3, and SETD1A), while two patients carried an inherited VUS. In addition, P/LP CNVs were observed in 3/15 patients (involving SATG2, KIAA0442, and MEIS2). CONCLUSIONS: All nine detected P/LP variants involved genes already known to lead to syndromic ID/DD; however, for only two genes (EHMT1 and MYT1L) is the link with obesity well established. This is the first study applying a comprehensive genomic investigation of an SO cohort, showing a high diagnostic yield (~47%). Additionally, our findings suggested that several known ID/DD genes may also predispose individuals to SO.

High frequency of genetic/epigenetic disorders in short stature children born with very low birth weight.

Most infants born with very low birth weight (VLBW, birth weight < 1500 g) show spontaneous catch-up growth in postnatal life. The reasons for the absence of catch-up growth are not entirely understood. We performed a comprehensive investigation of 52 children born with VLBW. Ten children had a history of an external cause that explained the VLBW and five refused genetic evaluation. Twenty-three cases were initially evaluated by a candidate gene approach. Patients with a negative result in the candidate gene approach (n = 14) or without clinical suspicion (n = 14) were assessed by chromosome microarray analysis (CMA) and/or whole-exome sequencing (WES). A genetic condition was identified in 19 of 37 (51.4%) patients without an external cause, nine by candidate gene approach, and 10 by a genomic approach (CMA/WES). Silver-Russell syndrome was the most frequent diagnosis (n = 5) and the remaining patients were diagnosed with other rare monogenic conditions. Almost all patients with a positive genetic diagnosis exhibited syndromic features (94.4%). However, microcephaly, neurodevelopmental disorders, major malformation, or facial dysmorphism were also frequently observed in children with an external cause. In conclusion, a significant proportion of children born with VLBW with persistent short stature have a genetic/epigenetic condition.

Insights from the genetic characterization of central precocious puberty associated with multiple anomalies.

STUDY QUESTION: Is there an (epi)genetic basis in patients with central precocious puberty (CPP) associated with multiple anomalies that unmasks underlying mechanisms or reveals novel genetic findings related to human pubertal control? SUMMARY ANSWER: In a group of 36 patients with CPP associated with multiple phenotypes, pathogenic or likely pathogenic (epi)genetic defects were identified in 12 (33%) patients, providing insights into the genetics of human pubertal control. WHAT IS KNOWN ALREADY: A few studies have described patients with CPP associated with multiple anomalies, but without making inferences on causalities of CPP. Genetic-molecular studies of syndromic cases may reveal disease genes or mechanisms, as the presentation of such patients likely indicates a genetic disorder. STUDY DESIGN, SIZE, DURATION: This translational study was based on a genetic-molecular analysis, including genome-wide high throughput methodologies, for searching structural or sequence variants implicated in CPP and DNA methylation analysis of candidate regions. PARTICIPANTS/MATERIALS, SETTING, METHODS: A cohort of 197 patients (188 girls) with CPP without structural brain lesions was submitted to a detailed clinical evaluation, allowing the selection of 36 unrelated patients (32 girls) with CPP associated with multiple anomalies. Pathogenic allelic variants of genes known to cause monogenic CPP (KISS1R, KISS1, MKRN3 and DLK1) had been excluded in the entire cohort (197 patients). All selected patients with CPP associated with multiple anomalies (n = 36) underwent methylation analysis of candidate regions and chromosomal microarray analysis. A subset (n = 9) underwent whole-exome sequencing, due to presenting familial CPP and/or severe congenital malformations and neurocognitive abnormalities. MAIN RESULTS AND THE ROLE OF CHANCE: Among the 36 selected patients with CPP, the more prevalent associated anomalies were metabolic, growth and neurocognitive conditions. In 12 (33%) of them, rare genetic abnormalities were identified: six patients presented genetic defects in loci known to be involved with CPP (14q32.2 and 7q11.23), whereas the other six presented defects in candidate genes or regions. In detail, three patients presented hypomethylation of DLK1/MEG3:IG-DMR (14q32.2 disruption or Temple syndrome), resulting from epimutation (n = 1) or maternal uniparental disomy of chromosome 14 (n = 2). Seven patients presented pathogenic copy number variants: three with de novo 7q11.23 deletions (Williams-Beuren syndrome), three with inherited Xp22.33 deletions, and one with de novo 1p31.3 duplication. Exome sequencing revealed potential pathogenic variants in two patients: a sporadic female case with frameshift variants in TNRC6B and AREL1 and a familial male case with a missense substitution in UGT2B4 and a frameshift deletion in MKKS. LIMITATIONS, REASONS FOR CAUTION: The selection of patients was based on a retrospective clinical characterization, lacking a longitudinal inclusion of consecutive patients. In addition, future studies are needed, showing the long-term (mainly reproductive) outcomes in the included patients, as most of them are not in adult life yet. WIDER IMPLICATIONS OF THE FINDINGS: The results highlighted the relevance of an integrative clinical-genetic approach in the elucidation of mechanisms and factors involved in pubertal control. Chromosome 14q32.2 disruption indicated the loss of imprinting of DLK1 as a probable mechanism of CPP. Two other chromosomal regions (7q11.23 and Xp22.33) represented new candidate loci potentially involved in this disorder of pubertal timing. STUDY FUNDING/COMPETING INTEREST(S): This work was supported by grant number 2018/03198-0 (to A.P.M.C.) and grant number 2013/08028-1 (to A.C.V.K) from the São Paulo Research Foundation (FAPESP), and grant number 403525/2016-0 (to A.C.L.) and grant number 302849/2015-7 (to A.C.L.) and grant number 141625/2016-3 (to A.C.V.K) from the National Council for Scientific and Technological Development (CNPq). The authors have nothing to disclose. TRIAL REGISTRATION NUMBER: N/A.

Noonan syndrome patients beyond the obvious phenotype: A potential unfavorable metabolic profile.

Noonan syndrome (NS) and NS related disorders (NRD) are frequent monogenic diseases. Pathogenic variants in PTPN11 are observed in approximately 50% of these NS patients. Several pleiotropic phenotypes have previously been described in this condition. This study aimed at characterizing glucose and lipid profiles in patients with NS/NRD. We assessed fasting blood glucose, insulin, cholesterol (total and fractions), and triglyceride (TG) levels in 112 prepubertal children and 73 adults. Additionally, an oral glucose tolerance test (OGTT) was performed in 40 children and 54 adults. Data were analyzed between age groups according to the presence (+) or absence (-) of PTPN11 mutation. Prepubertal patients with NS/NRD were also compared with a control group. Despite the lean phenotype of children with NS/NRD, they presented an increased frequency of low HDL-cholesterol (63% in PTPN11+, 59% in PTPN11- and 16% in control, p < .001) and high TG levels (29% in PTPN11+, 18% in PTPN11- and 2.3% in control). PTPN11+ patients had a higher median HOMA-IR (1.0, ranged from 0.3 to 3.2) in comparison with PTPN11- (0.6; 0.2 to 4.4) and controls (0.6; 0.4 to 1.4, p = .027). Impaired glucose tolerance was observed in 19% (10:54) of lean adults with NS/NRD assessed by OGTT. Moreover, women with PTPN11 mutations had lower HDL-cholesterol levels than those without. Our results suggest that children and young adult patients with NS/NRD have an unfavorable metabolic profile characterized by low HDL, a tendency of elevated TGs, and glucose metabolism impairment despite a lean phenotype.

Congenital chromoanagenesis in the routine postnatal chromosomal microarray analyses.

Chromosomal microarray analyses (CMA) have greatly increased both the yield and diagnostic accuracy of postnatal analysis; it has been used as a first-tier cytogenetic test in patients with intellectual disability, autism spectrum disorder, and multiple congenital abnormalities. During the last 15 years, we performed CMA in approximately 8,000 patients with neurodevelopmental and/or congenital disorders, of which 13 (0.16%) genetically catastrophic complex chromosomal rearrangements were identified. These ultrarare rearrangements showed clustering of breakpoints, characteristic of chromoanagenesis events. Al1 13 complex events display underlying formation mechanisms, originating either by a synchronization of the shattering of clustered chromosome regions in which regional asynchrony of DNA replication may be one of the main causes of disruption. We provide an overview of the copy number profiling in these patients. Although several previous studies have suggested that chromoanagenesis is often a genetic disease source in postnatal diagnostic screening, due to either the challenge of clinical interpretation of these complex rearrangements or the limitation of microarray resolution relative to the small size and complexity of chromogenic induced chromosome abnormalities, bringing further attention and to study its occurrence in the clinical setting is extremely important.

Genetics, clinical features and outcomes of non-syndromic pituitary gigantism: experience of a single center from Sao Paulo, Brazil.

PURPOSE: Non-syndromic pituitary gigantism (PG) is a very rare disease. Aryl hydrocarbon receptor-interacting protein (AIP) and G protein-coupled receptor 101 (GPR101) genetic abnormalities represent important etiologic causes of PG and may account for up to 40% of these cases. Here, we aimed to characterize the clinical and molecular findings and long-term outcomes in 18 patients (15 males, three females) with PG followed at a single tertiary center in Sao Paulo, Brazil. METHODS: Genetic testing for AIP and GPR101 were performed by DNA sequencing, droplet digital PCR and array comparative genomic hybridization (aCGH). RESULTS: Pathogenic variants in the AIP gene were detected in 25% of patients, including a novel variant in splicing regulatory sequences which was present in a sporadic male case. X-LAG due to GPR101 microduplication was diagnosed in two female patients (12.5%). Of interest, these patients had symptoms onset by age 5 and 9 years old and diagnosis at 5 and 15 years, respectively. X-LAG, but not AIP, patients had a significantly lower age of symptoms onset and diagnosis and a higher height Z-score when compared to non-X-LAG. No other differences in clinical features and/or treatment outcomes were observed among PG based on their genetic background. CONCLUSION: We characterize the clinical and molecular findings and long-term outcome of the largest single-center PG cohort described so far.

Phenotype-genotype analysis of 242 individuals with RASopathies: 18-year experience of a tertiary center in Brazil.

We report the clinical and molecular data of a large cohort comprising 242 individuals with RASopathies, from a single Tertiary Center in Brazil, the largest study from Latin America. Noonan syndrome represented 76% of the subjects, with heterozygous variants in nine different genes, mainly PTPN11, SOS1, RAF1, LZTR1, and RIT1, detected by Sanger and next-generation sequencing. The latter was applied to 126 individuals, with a positive yield of 63% in genes of the RAS/MAPK cascade. We present evidence that there are some allelic differences in PTPN11 across distinct populations. We highlight the clinical aspects that pose more medical concerns, such as the cardiac anomalies, bleeding diathesis and proliferative lesions. The genotype-phenotype analysis between the RASopathies showed statistically significant differences in some cardinal features, such as craniofacial and cardiac anomalies, the latter also statistically significant for different genes in Noonan syndrome. We present two individuals with a Noonan syndrome phenotype, one with an atypical, structural cardiac defect, harboring variants in genes mainly associated with isolated hypertrophic cardiomyopathy and discuss the role of these variants in their phenotype.

Clinical and Genetic Characterization of a Constitutional Delay of Growth and Puberty Cohort.

INTRODUCTION: Constitutional delay of growth and puberty (CDGP) is the most prevalent cause of delayed puberty in both sexes. Family history of delayed puberty (2 or more affected members in a family) has been evidenced in 50-75% of patients with CDGP and the inheritance is often consistent with autosomal dominant pattern, with or without complete penetrance. However, the molecular basis of CDGP is not completely understood. OBJECTIVE: To characterize the clinical and genetic features of a CDGP cohort. METHODS: Fifty-nine patients with CDGP (48 boys and 11 girls) underwent careful and long-term clinical evaluation. Genetic analysis was performed using a custom DNA target enrichment panel designed to capture 36 known and candidate genes implicated with pubertal development. RESULTS: All patients had spontaneous or induced pubertal development (transient hormonal therapy) prior to 18 years of age. The mean clinical follow-up time was 46 ± 28 months. Male predominance (81%), short stature (91%), and family history of delayed puberty (59%) were the main clinical features of this CDGP -cohort. Genetic analyses revealed 15 rare heterozygous missense variants in 15 patients with CDGP (25%) in seven different genes (IGSF10, GHSR, CHD7, SPRY4, WDR11, SEMA3A,and IL17RD). IGSF10 and GHSR were the most prevalent affected genes in this group. CONCLUSIONS: Several rare dominant variants in genes implicated with GnRH migration and metabolism were identified in a quarter of the patients with familial or sporadic CDGP, suggesting genetic heterogeneity in this frequent pediatric condition.

Selective Tryptophan Oxidation of Monoclonal Antibodies: Oxidative Stress and Modeling Prediction.

Oxidation of tryptophan not only generates heterogeneity of a therapeutic monoclonal antibody (mAb) but also can be a potential critical quality attribute (CQA) of the product. In this study, mAbs A-C of IgG1 and IgG4 (immunoglobulin G, IgG) isotypes with oxidized tryptophan (Trp) residues were selectively generated by incubating the mAbs with 2,2'-azobis(2-amidinopropane) dihydrochloride (AAPH) in formulations containing l-methionine. The site-specific oxidation of tryptophan residues were confirmed by liquid chromatography coupled with mass spectrometry (LC-MS) studies. The site of oxidation was identified to be a conserved tryptophan residue in the heavy chain complementarity determining region 3 (CDR3) of mAbs A and B with no significant oxidation found on other tryptophan residues including those in close proximity to CDR3. For mAb C, all tryptophan residues including one in the heavy chain CDR1 and a tryptophan in close proximity to heavy chain CDR3 were not susceptible to oxidation. For all three mAbs, the structure and tryptophan oxidation relationship was further studied by computational modeling of the variable domain of the antibodies (variable fragment, Fv). The computational modeling provided a structural understanding at the molecular level to the tryptophan oxidation, where high solvent accessibility is a prerequisite for heavy chain CDR3 tryptophan oxidation. However, higher oxidation susceptibility of tryptophan in heavy chain CDR3 did not linearly correlate to higher solvent accessibility, suggesting that other factors including side-chain orientation and/or surrounding structure elements around the heavy chain CDR3 may also be involved. Through this study, we demonstrate that a selective oxidation system, together with computational modeling, can be an important tool to identify potential CQAs of a therapeutic mAb such as tryptophan oxidation liabilities during the mAb's development.

Genetic Disorders in Prenatal Onset Syndromic Short Stature Identified by Exome Sequencing.

OBJECTIVE: To perform a prospective genetic investigation using whole exome sequencing of a group of patients with syndromic short stature born small for gestational age of unknown cause. STUDY DESIGN: For whole exome sequencing analysis, we selected 44 children born small for gestational age with persistent short stature, and additional features, such as dysmorphic face, major malformation, developmental delay, and/or intellectual disability. Seven patients had negative candidate gene testing based on clinical suspicion and 37 patients had syndromic conditions of unknown etiology. RESULTS: Of the 44 patients, 15 (34%) had pathogenic/likely pathogenic variants in genes already associated with growth disturbance: COL2A1 (n = 2), SRCAP (n = 2), AFF4, ACTG1, ANKRD11, BCL11B, BRCA1, CDKN1C, GINS1, INPP5K, KIF11, KMT2A, and POC1A (n = 1 each). Most of the genes found to be deleterious participate in fundamental cellular processes, such as cell replication and DNA repair. CONCLUSIONS: The rarity and heterogeneity of syndromic short stature make the clinical diagnosis difficult. Whole exome sequencing allows the diagnosis of previously undiagnosed patients with syndromic short stature.

Evaluation of SHOX defects in the era of next-generation sequencing.

Short stature homeobox (SHOX) haploinsufficiency is a frequent cause of short stature. Despite advances in sequencing technologies, the identification of SHOX mutations continues to be performed using standard methods, including multiplex ligation-dependent probe amplification (MLPA) followed by Sanger sequencing. We designed a targeted panel of genes associated with growth impairment, including SHOX genomic and enhancer regions, to improve the resolution of next-generation sequencing for SHOX analysis. We used two software packages, CONTRA and Nexus Copy Number, in addition to visual analysis to investigate the presence of copy number variants (CNVs). We evaluated 15 patients with previously known SHOX defects, including point mutations, deletions and a duplication, and 77 patients with idiopathic short stature (ISS). The panel was able to confirm all known defects in the validation analysis. During the prospective evaluation, we identified two new partial SHOX deletions (one detected only by visual analysis), including an intragenic deletion not detected by MLPA. Additionally, we were able to determine the breakpoints in four cases. Our results show that the designed panel can be used for the molecular investigation of patients with ISS, and it may even detect CNVs in SHOX and its enhancers, which may be present in a significant fraction of patients.

BCL11B mutations in patients affected by a neurodevelopmental disorder with reduced type 2 innate lymphoid cells.

The transcription factor BCL11B is essential for development of the nervous and the immune system, and Bcl11b deficiency results in structural brain defects, reduced learning capacity, and impaired immune cell development in mice. However, the precise role of BCL11B in humans is largely unexplored, except for a single patient with a BCL11B missense mutation, affected by multisystem anomalies and profound immune deficiency. Using massively parallel sequencing we identified 13 patients bearing heterozygous germline alterations in BCL11B. Notably, all of them are affected by global developmental delay with speech impairment and intellectual disability; however, none displayed overt clinical signs of immune deficiency. Six frameshift mutations, two nonsense mutations, one missense mutation, and two chromosomal rearrangements resulting in diminished BCL11B expression, arose de novo. A further frameshift mutation was transmitted from a similarly affected mother. Interestingly, the most severely affected patient harbours a missense mutation within a zinc-finger domain of BCL11B, probably affecting the DNA-binding structural interface, similar to the recently published patient. Furthermore, the most C-terminally located premature termination codon mutation fails to rescue the progenitor cell proliferation defect in hippocampal slice cultures from Bcl11b-deficient mice. Concerning the role of BCL11B in the immune system, extensive immune phenotyping of our patients revealed alterations in the T cell compartment and lack of peripheral type 2 innate lymphoid cells (ILC2s), consistent with the findings described in Bcl11b-deficient mice. Unsupervised analysis of 102 T lymphocyte subpopulations showed that the patients clearly cluster apart from healthy children, further supporting the common aetiology of the disorder. Taken together, we show here that mutations leading either to BCL11B haploinsufficiency or to a truncated BCL11B protein clinically cause a non-syndromic neurodevelopmental delay. In addition, we suggest that missense mutations affecting specific sites within zinc-finger domains might result in distinct and more severe clinical outcomes.

Mutations in C-natriuretic peptide (NPPC): a novel cause of autosomal dominant short stature.

PurposeC-type natriuretic peptide (CNP) and its principal receptor, natriuretic peptide receptor B (NPR-B), have been shown to be important in skeletal development. CNP and NPR-B are encoded by natriuretic peptide precursor-C (NPPC) and natriuretic peptide receptor 2 (NPR2) genes, respectively. While NPR2 mutations have been described in patients with skeletal dysplasias and idiopathic short stature (ISS), and several Npr2 and Nppc skeletal dysplasia mouse models exist, no mutations in NPPC have been described in patients to date.MethodsNPPC was screened in 668 patients (357 with disproportionate short stature and 311 with autosomal dominant ISS) and 29 additional ISS families in an ongoing whole-exome sequencing study.ResultsTwo heterozygous NPPC mutations, located in the highly conserved CNP ring, were identified. Both showed significant reductions in cyclic guanosine monophosphate synthesis, confirming their pathogenicity. Interestingly, one has been previously linked to skeletal abnormalities in the spontaneous Nppc mouse long-bone abnormality (lbab) mutant.ConclusionsOur results demonstrate, for the first time, that NPPC mutations cause autosomal dominant short stature in humans. The NPPC mutations cosegregated with a short stature and small hands phenotype. A CNP analog, which is currently in clinical trials for the treatment of achondroplasia, seems a promising therapeutic approach, since it directly replaces the defective protein.